Biotreatment of phenol by Rhodococcus sp.GM3 in

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Biotreatment of phenol by Rhodococcus sp.GM3 in packed-bed bioreactor Mahammed E Jabbar, K.Kiran Kumar , Bee Hameeda and Gopal Reddy Department of Microbiology, Osmania University, Hyderabad, Andhra Pradesh, India-500 007 Email: [email protected]

ABSTRACT Biotreatment is use of living organisms for removal of a pollutant from biosphere. Phenol is one of the major organic pollutants present among a wide variety of highly toxic organic chemicals. Continuous phenol degradation experiment was carried out in a packedbed column bioreactor. A cylindrical glass column with internal diameter of 4.0 cm was filled with Ca-alginate immobilized beads and polyurethane foam encapsulated with Rhodococcus sp.GM3 to a height of 16 cm separately. The column was attached to a reservoir of sterile 250 mL mineral salts medium adjusted to pH 8.5 containing different concentrations of phenol (0.5, 1.0, 1.5 and 2.0 g/L). Phenol was degraded in Ca-alginate immobilized and polyurethane foam on packed bed reactors with different concentrations and the maximum effective rate of phenol degradation was observed to be 83.33 mg/L per hour, whereas the flow rate of 1.0 mL/sec was efficient for degradation of phenol. Keywords : Biotreatment, Bioreactor, Immobilized cells, Phenol Introduction Biotreatment of xenobiotic pollutants by microbial communities remains a major challenge to microbial ecologists, out of which phenol is a major pollutant present in the wastewater from various industrial activities such as pharmaceutical, polymeric resin production, refineries, petrochemicals, petroleum, chemical pulp and paper mills, coking operations, coal refining, stainless steel production, textile, tannery and foundries (Santos and Linardi, 2001; Nor Suhaila et al., 2010; Basha et al., 2010). Phenol is an important recalcitrant pollutant and considered as primary contaminant in waste water due to slow biodegradability and high toxicity. When phenol-bearing water

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is chlorinated, toxic polychlorinated phenols can be formed. United State Environmental Protection Agency (USEPA) has thus set a water purification standard of phenol less than 1.0 mg/L in surface waters (Chung et al., 2003). Phenol is an important organic pollutant and is a primary contaminant in wastewater due to its toxicity to mammals (van Agteren et al., 1998), the minimum reported lethal oral dose in humans was approximately 70 mg/kg-body weight. Phenol is highly irritating to the skin, eyes, and mucous membranes. Systemic effects in humans include gastrointestinal irritation, dermal necrosis, and cardiac arrhythmias. Lethal concentrations of phenol produce symptoms of muscle weakness, convulsions, and coma (Campbell, 2003). Many technologies were applied for the abatement of phenol from water streams such as membrane-solvent extraction, ozonation, biochemical treatments, electrochemical and photocatalytic oxidation, condensation, absorption in liquids, adsorption on solids, membrane separation etc. (Busca et al., 2008), but the remediation efficiency of xenobiotic pollutants by microbial cells remains a major challenge to microbial ecologists and process engineers (Whiteley and Bailey, 2000), Notably the biological treatment is a more preferable alternative to traditional physical and chemical treatment methods due its low cost, reliable operational stability and highly efficient control of pollution. It is necessary to exploit these bacteria for removal of phenol from any waste before it reaches the environment. Thus the aim of this study is to treat phenol by continuous biodegradation experiments in packed bed reactor that is filled with Ca-alginate immobilized beads and polyurethane foam separately, immobilized ability of Rhodococcus sp. GM3 to degrade phenol was studied at two flow rates and different influent phenol concentrations, also artificial wastewater was used with different concentrations of phenol. Material and Methods Isolation and identification Samples were collected from 4 industrial and 4 agricultural sites in Hyderabad. Enrichment of phenol degrading bacteria was carried out to screen 8 soil samples. One of the bacterial isolate from a total of 26 isolates showed high phenol degradation and it has been identified as Rhodococcus sp. GM3. Phenol estimation Direct photometric method (Clesceri et al., 1998) was used for estimation of phenol concentration by adding 4-amino-antipyrene at pH 7.9 ± 0.1 using ammonium hydroxide

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and phosphate buffer, followed by oxidation with alkaline K 3Fe(CN)6 giving a red color when phenol is present. To estimate the phenol, left over samples were collected from a outlet of the packed bed column and centrifuged at 5000 rpm for 10 minutes to remove cell pellet and was analyzed by UV/visible recording spectrophotometer SHIMADZU 160A (Tokyo, Japan) at 500 nm. Inoculum preparation for immobilization A 250 mL conical flask containing 50 mL mineral salts medium (MSM) broth (pH 8.5) was inoculated using a fresh slant culture, MSM broth inoculated with Rhodococcus sp.GM3 was incubated in a shaker incubator at 200 rpm, temperature 32ºC. A 16 hr culture (exponentially grown) is harvested by centrifuging at 4000 rpm for 5 minutes in a Sigma cold centrifuge at 4ºC in 100 mL sterile tubes. The cell pellet was washed with sterile normal saline twice and the cell pellet was suspended in 0.1 M phosphate buffer ( Nagavalli, 2009). Alginate entrapment of cells Sodium alginate and calcium chloride were used to prepare the alginate beads containing the whole cells. Sodium alginate solution was prepared by dissolving 3g of sodium alginate in 90 mL hot distilled water, stirred vigorously for 10 mins to obtain thick uniform slurry without any undissolved clumps. The alginate slurry and cell suspension each 1 mL containing 45×109 bacteria were mixed for 8 mins at ratio 10% (v/v) to get uniform mixture with a final concentration of 3% (w/v) sodium alginate. The slurry was taken into sterile syringe and then dropped drop wise into ice cold 0.2 M CaCl 2.2H2O solution from 5 cm height and kept for curing at 4ºC for 1 hour. The beads were washed with sterile saline solution and refrigerated for further use in experiments. All these operation were carried out aseptically under laminar air flow unit. Polyurethane foam (PUF) entrapment of cells A PUF (0.5 cm ×0.5 cm ×0.5 cm) placed in flask after autoclaving at 121 ºC for 20 min, 2 mL of inoculum was added to 250mL flask containing 50mL of MSM incubation at 32ºC and 75 rpm for 6 days. Enumeration of viable cell was carried out 6 days after inoculation. To ensure that only the immobilized cells were quantified, the PUF was first rinsed with sterile MSM. The PUF was transferred into the column using sterile forceps.

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Packed bed reactor This experiment was conducted to check for continuous degradation of phenol using a packed - bed column. The packed bed column described here consists of a cylindrical glass column of 16.0 cm height and 4.0 cm internal diameter. This cylindrical column was filled with bacterial cells immobilized in Ca- alginate and polyurethane foam, glass wool was placed at the bottom of the column to pack the immobilized beads in the glass column. For uniform distribution of media a porous glass frit was placed on the top layer of the beads in the column and a sample collecting port was arranged for regular collection of the samples. A 500 mL flask containing 250 mL of sterile MSM (pH 8.5) was attached to the column which acts as feed reservoir and experiments were conducted with varying phenol concentrations (0.5, 1.0, 1.5 and 2.0 g/L). The medium was then fed in to the column continuously using a dosing pump (FTPS model FW 04 05) down flow the experiments were conducted at two flow rates (0.5 and 1.0 mL/sec) at temperature of 30 – 35°C. During the continuous operation, media containing phenol was pumped and samples were collected at different time intervals, centrifuged at 5000 rpm for 10 min, phenol was estimated in the supernatant. The schematic view of continuous flow of packed bed reactor is depicted in Figure (1) To study the application of phenol degrading bacteria simulated wastewater (Martin et al., 2000) was used at flow rates 1.0 mL/sec with phenol concentrations (1.5 and 2.0 g/ L). The artificial wastewater (ASW) prepared in distilled water containing (per liter) 40 mg of K2HPO4, 10 mg of KH2PO4, 50 mg of (NH4)2SO4, 25 mg of KNO3, 25 mg of MgSO4 .7 H2O, 2 mg of FeSO4 .7 H2O and 10 mg of CaSO4 (pH 8.5). Results Packed bed reactor is a cylindrical vessel in which degradation process was carried out using Rhodococcus sp. GM3, the medium (MSM or ASW) was then fed into the column continuously containing different concentrations of phenol. The bioreactor was maintained as a closed system with no air sparging to eliminate the substrate loss due to volatility. The time required for degradation of 0.5, 1.0, 1.5 and 2.0 g/L phenol by Ca-alginate immobilized beads were 9, 12, 18 and 24 hours respectively for both flow rates used (Figure 2). The time required for degradation of 0.5, 1.0, 1.5 and 2.0 g/L phenol by PUF immobilized cells at flow rate of 0.5 mL/sec were 9, 12, 24 and 24 hours respectively and at flow rate of 1.0 mL/sec were 6, 9, 18 and 24 hours respectively (Figure .3) Thereby the rate of phenol degradation in MSM by Ca-alginate immobilized beads

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was 83.33 mg/L per hour of phenol (1.0, 1.5 and 2.0 g/L) at flow rate 0.5 and 1.0 mL/sec while the rate of phenol degradation decreased at phenol feed concentration 0.5g/L than other concentrations. The rate of phenol degradation on MSM by polyurethane foam immobilized cells was 83.33 mg/L per hour of all phenol concentrations at flow rate 1.0 mL/sec (Table 1). Further biodegradation studies at phenol concentrations in artificial wastewater, the result showed 1.5 g/L phenol was completely degraded by Ca-alginate and PUF immobilized within 24 hours and 2.0 g/L within 30 and 36 hours by Ca-alginate and PUF immobilized cells respectively, as obvious from the data presented in Figure 4. The rate of phenol degradation on ASW was fairly less than MSM by Ca-alginate and PUF immobilized cells (Table 2). However these result suggest that the application of immobilized cells in wastewater treatment offers the possibility of degrading higher concentrations of phenol that can be achieved by bioreactor, although phenol is considered to be inhibitory and toxic to microorganisms. According to these results, Ca-alginate and polyurethane foam immobilized can be used for effective phenol degradation, however PUF immobilized at a flow rate of 1.0 mL/ sec ascertains more rate of phenol degradation. Discussion The results indicated that phenol removal efficiency ranging from 0.5 to 1.5 g/L at a rate maximum of 83.33 mg/L per hour of phenol by Ca-alginate immobilized beads and PUF immobilized on MSM, hence these demonstrated the ability of the packed bed reactor to remove the phenol with using a variety of packing materials, also increase in the flow rate to keep aerobic conditions along the entire bed height by allowing the dissolved oxygen in the outflow. Tziotzios et al. (2005) reported the maximum phenol removal rate reached only 82 mg/L per day for phenol feed concentration 0.512 g/L, while Prieto et al.( 2002) observed a maximum removal rate of 2.2 g/d for feed phenol concentration of 0.2 g /L using cultures of Rhodococcus erythropolis in 150 mL glass columns filled with Biolite beads. Considerably more favorable for degradation is the potential of immobilizing microorganisms onto (PUF), alginate and other matrices to degrade hydrocarbons and toxic waste. Quek et al. (2006) suggested the potential of using PUF-immobilized Rhodococcus sp. F92 to bioremediate petroleum hydrocarbons in an open marine environment Eugene. In the present study, an immobilized packed bed reactor design was used for the continuous degradation of phenol from media and wastewater. The result clearly indicates the packed bed reactor can degrade phenol on artificial wastewater containing 1.5 and 2.0

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g/L phenol when filled with immobilized Rhodococcus sp. GM3 in Ca-alginate and PUF. Guo (2010) reported the strain had strong adaptability to the environmental conditions, which showed it was a high effective phenol degradation strain and had better application prospects in wastewater treatment. Increase of feed phenol concentration into packed bed reactors on the biological degradation of phenol to about 2750 mg/L demonstrated inhibitory effects on bacterial growth (Tziotzios et al., 2007). Rhodococcus spp. are increasingly becoming more important in the field of bioremediation and biotechnology due to their ability to degrade many pollutants and to produce biosurfactants or emulsifiers with beneficial applications (Bell et al., 1998). The data revealed that at flow rate of 1.0 mL/sec showed more influence for phenol degradation than 0.5 mL/sec. Yet both these rates correspond well in phenol degradation, it is known that sufficient supply of oxygen is a critical factor in aerobic bioprocesses. On the contrary, the removal efficiency in the reactor decreased with an increase in the liquid flow rate inlet (Lin et al., 2009). Attention focused on immobilized cell reactors achieved high degradation with better process stability, but they require pumping motor and tools therefore are costly to operate. A proper choice of immobilized culture, careful consideration of various design parameters for a semifluidized bed bioreactor will make treatment process cost effective in the long run (Meikap and Rot, 1997). In addition to technical advantages, the application of immobilized cells in wastewater treatment offers the possibility of tolerating higher concentrations of toxic pollutants. Several studies demonstrate that immobilized microorganisms are better protected against phenolic compounds than the free-cells in the media (Keweloh et al., 1989; Kim et al., 2006) Although wastewaters containing phenols are often problematic because of the toxicity and recalcitrance of phenol compounds (Yotova et al., 2009), however; difficulties arise in such treatment due to the toxicity of phenol to the microbial population (Loh et al., 2000) but fitting results obtained in this work demonstrated the ability of the lab-scale packed bed reactor to degrade the phenol using different packing materials (Alginate and PUF) tested by immobilized Rhodococcus sp. GM3. Conclusion It is deduced that different concentrations of phenol were degraded in MSM when Ca-alginate and PUF immobilized cells were used in packed-bed reactor at flow rate 0.5 and 1.0 mL/sec and maximum rate of phenol was 83.33 mg/L per hour. Also the data suggest that the flow rate of 1.0 mL/sec showed more influence for phenol degradation than 0.5 mL/sec. Phenol is major pollutant, discharged from many industrial process, therefore requires

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proper treatment before being discharged to environment, the packed-bed reactor can degrade phenol on artificial wastewater containing 1.5 and 2.0 g/L phenol when filled with immobilized Rhodococcus sp. GM3 Ca-alginate and PUF. Acknowledgements The author (Md E Jabbar) thanks the Indian Council for Cultural Relations (ICCR), Govt. of India for providing the fellowship to carry out this research work. References 1.

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Fig 1: Schematic representation of the packed bed reactor

Fig 2: Phenol degradation in packed bed reactor with Ca-alginate immobilized Rhodococcus sp. GM3 in mineral salts medium containing 0.5, 1.0, 1.5 and 2.0 g/L phenol concentration.

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Fig: 3 Phenol degradation in packed bed reactor with PUF immobilized Rhodococcus sp. GM3 in mineral salts medium containing 0.5, 1.0, 1.5 and 2.0 g/L phenol concentration.

Fig: 4 Phenol degradation in packed bed reactor with Ca-alginate and PUF Rhodococcus sp. GM3 immobilized in artificial wastewater containing 1.5 and 2.0 g/L phenol concentration.

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Table 1: Rate of Phenol degradation in packed bed reactor with Ca-alginate and PUF immobilized Rhodococcus sp. GM3 in mineral salts medium containing 0.5, 1.0, 1.5 and 2.0 g/L phenol concentration. Initial phenol concentrations g/L

Rate of phenol degradation mg/L per hour Ca-alginate

Polyurethane foam

Flow rate 0.5 mL/sec

Flow rate 1.0 mL/sec

Flow rate 0.5 mL/sec

55.55 66.66 83.33 83.33

55.55 83.33 83.33 83.33

55.55 83.33 62.5 83.33

0.5 1.0 1.5 2.0

Flow rate 1.0 mL/sec 83.33 83.33 83.33 83.33

Table 2: Rate of Phenol degradation in packed bed reactor with Ca-alginate and PUF immobilized Rhodococcus sp. GM3 in artificial wastewater containing 1.5 and 2.0 g/L phenol concentration. Initial phenol

Rate of phenol degradation mg/L per hour

concentrations g/L

Ca-alginate

1.5 2.0

62.5 55.55

Polyurethane foam 62.5 66.66